Microfluidic device with multiple microcoil NMR detectors enabling fluidic series communication

ABSTRACT

An NMR system comprises an NMR probe comprising multiple NMR detection sites. Each of the multiple NMR detection sites comprises a sample holding void and an associated NMR microcoil. The NMR system further comprises a controllable fluid router operative to direct fluid sample to the multiple NMR detection sites.

PRIORITY

This application is a continuation of abandoned U.S. patent applicationSer. No. 10/852,672, filed May 24, 2004, which corresponds to PatentApplication Publication U.S. 2005/0030033 A1 published Feb. 10, 2005,which was a continuation of U.S. patent application Ser. No. 10/003,837,filed Dec. 3, 2001, which corresponds to Patent Application Publication2002/0149369 A1 published Oct. 17, 2002 and is now U.S. Pat. No.6,822,454 B2 which issued on Nov. 23, 2004 and which claims the prioritybenefit of Provisional Patent Application No. 60/250,874 filed Dec. 1,2000.

FIELD OF THE INVENTION

The present invention is directed to microfluidic devices havingmultiple microcoil nuclear magnetic resonance (NMR) detectors and, moreparticularly to microfluidic devices having improved microcoil NMRdetectors for capillary-scale, high resolution NMR spectroscopy probescapable of enhanced sample processing functionality.

BACKGROUND

Nuclear magnetic resonance spectroscopy, or NMR, is a powerful andcommonly used method for analysis of the chemical structure ofmolecules. NMR provides spectral information as a function of theelectronic environment of the molecule and is nondestructive to thesample. In addition, reaction rates, coupling constants, bond-lengths,and two- and three-dimensional structure can be obtained with thistechnique.

Systems for biochemical, chemical, and molecular analysis can beminiaturized as capillary-based systems or substrate-based, i.e.,micro-scale, systems with multifunctional capabilities including, forexample, chemical, optical, fluidic, electronic, acoustic, and/ormechanical functionality. Miniaturization of these systems offersseveral advantages, including increased complexity, functionality, andefficiency. Devices can be fabricated from diverse materials including,for example, plastics, polymers, metals, silicon, ceramics, paper, andcomposites of these and other materials. Mesoscale sample preparationdevices for providing microscale test samples are described in U.S. Pat.No. 5,928,880 to Wilding et al. Devices for analyzing a fluid sample,comprising a solid substrate microfabricated to define at least onesample inlet port and a mesoscale flow channel extending from the inletport within the substrate for transport of a fluid sample are describedin U.S. Pat. No. 5,304,487. Currently known miniaturized fluid-handlingand detection devices have not met all of the needs of industry.

NMR is one of the most information-rich forms of biochemical, chemical,and molecular detection and analysis, and remains highly utilized in awide range of health-related industries, including pharmaceuticalresearch and drug discovery. One of the fundamental limitations of NMRfor these and other applications involves sample throughput. Whencompared to other forms of detection (e.g., mass spectrometry), theamount of sample required by NMR is generally orders of magnitudegreater, and correspondingly the mass limits of detection are generallyorders of magnitude poorer. Conventional NMR spectrometers typically userelatively large RF coils (mm to cm size) and samples in the ml volumerange, and significant performance advantages are achieved using NMRmicrocoils when examining very small samples. Prior to such developmentof microcoil NMR and NMR flowprobes, NMR remained a test tube-basedanalytical technique requiring milliliters of sample and often requiringdata acquisition times ranging from 10 min. to several hours forinformative spectra with sufficient signal to noise ratio (“S/N”). NMRmicrocoils are known to those skilled in the art and are shown, forexample, in U.S. Pat. No. 5,654,636 to Sweedler et al., and in U.S. Pat.No. 5,684,401 to Peck et al., and in U.S. Pat. No. 6,097,188 to Sweedleret al., all three of which patents are incorporated herein by referencein their entireties for all purposes. A solenoid microcoil detectioncell formed from a fused silica capillary wrapped with copper wire hasbeen used for static measurements of sucrose, arginine and other simplecompounds. Wu et al. (1994a), J. Am. Chem. Soc. 116:7929–7930; Olson etal. (1995), Science 270:1967–1970, Peck (1995) J. Magn. Reson. 108(B)114–124. Coil diameter has been further reduced by the use ofconventional micro-electronic techniques in which planar gold oraluminum R.F. coils having a diameter ranging from 10–200 .mu.m wereetched in silicon dioxide using standard photolithography. Peck 1994IEEE Trans Biomed Eng 41(7) 706–709, Stocker 1997 IEEE Trans Biomed Eng44(11) 1122–1127, Magin 1997 IEEE Spectrum 34 51–61.

Miniature total analysis systems (μ-TAS) are discussed in IntegratingMicrofluidic Systems And NMR Spectroscopy—Preliminary Results, Trumbullet al, Solid-State Sensor and Actuator Workshop, pp. 101–05 (1998),Magin 1997 IEEE Spectrum 34 51–61, and Trumbull 2000 47(1) 1–6. TheTrumbull et al. device integrated multiple chemical processing steps andthe means of analyzing their results on the same miniaturized system.Specifically, Trumbull et al. coupled chip-based capillaryelectrophoresis (CE) with nuclear magnetic resonance spectroscopy (NMR)in a μ-TAS system.

Capillary-based liquid chromatography and microcoil NMR have compatibleflow rates and sample volume requirements. Thus, for example, thecombination of the Waters CapLC™ available from Waters Corporation(Milford, Mass., USA) and the MRM CapNMR™ flow probe available from MRMCorporation (Savoy, Ill.), a division of Protasis Corporation(Marlborough, Mass., USA) provides excellent separation capability inaddition to UV-VIS and NMR detection for mass-limited samples. TheWaters CapLC™ has published flow rates from 0.02 μL/minute to 40μL/minute. A typical CapLC on-column flow rate is 5 μL/min, theautosampler-injected analyte volume is 0.1 μL or more, and accurate flowrates are achieved through capillary of typically 50 μm inner diameter.The NMR flow cell has a typical total volume of 5 μL with a microcoilobserve volume of 1 μL. A typical injected sample amount for CapLC-μNMRanalysis is a few μg (nmol) or less.

Capillary scale systems also are shown in U.S. Pat. No. 6,194,900, theentire disclosure of which is incorporated herein by reference for allpurposes. In such systems, a capillary-based analyte extraction chamberis connected to an NMR flow site, such as by being positioned as anoperation site along a capillary channel extending to the NMR flow cell.

Small volume flow probes are shown, for example, by Haner et al. inSmall Volume Flow Probe for Automated Direct-Injection NMR Analysis:Design and Performance, J. Magn. Reson., 143, 69–78 (2000).Specifically, Haner et al show a tubeless NMR probe employing anenlarged sample chamber or flowcell. Microcoil-based micro-NMRspectroscopy is disclosed in U.S. Pat. Nos. 5,654,636, 5,684,401, and6,097,188, the entire disclosures of all of which are incorporatedherein by reference for all purposes. Sample amounts can now range assmall as several hundred microliters for conventional flowprobes tosmaller than 1 uL for microcoil-based capillary-scale flowprobes.Acquisition times typically range from minutes to hours. The mostexpensive and technologically limiting component of the NMR system isthe superconducting magnet. Although significant financial and technicalinvestment has been made in the development of elaborate mechanical(robotic-controlled) sample changers and, more recently, automated flowinjection systems for repetitive and continuous sample throughput, themagnet remains today a dedicated component in which only sequential,one-at-a-time analysis of samples is carried out.

NMR is one of the few analytical methods in which parallel dataacquisition has not been applied to increase sample processingfunctionality, such as the number of samples that can be tested in agiven time. At least some of the difficulties in accomplishing thisobjective are intrinsically related to the hardware involved in NMR dataacquisition.

Recent academic results have shown that some of the limitations of NMRprocessing can be overcome by the use of multiple microcoil detectors ina wide-bore magnet. Proposed designs for incorporating multiplesolonoidal microcoils into a single probe head are presented by Li etal. in Multiple Solenoidal Microcoil Probes for High-Sensitivity,High-Throughput Nuclear Magnetic Resonance Spectroscopy, Anal. Chem.,71, 4815–4820, 1999. A dual channel probe for simultaneous acquisitionof NMR data from multiple samples is shown by Fisher et al. in NMR Probefor the Simultaneous Acquisition of Multiple Samples, J. Magn. Reson.,138, 160–163 (1999). Such devices, however, have not been commerciallyimplemented and have not been shown to be commercially viable. Inaddition, higher numbers of multiple microcoil detectors are needed thatare compatible also with narrow bore magnets, since narrow bore magnetsare predominant in industrial settings. There is also both need for andbenefit of microcoil NMR probes having enhanced sample processingfunctionality.

Accordingly, it is an object of the present invention to providemulti-microcoil NMR microfluidic devices having enhanced sampleprocessing functionality. It is a particular object of the invention toprovide improved microcoil NMR detectors for capillary-scale, highresolution NMR spectroscopy probes that can be adapted in accordancewith certain preferred embodiments for use in large or small boremagnets and that are capable of enhanced sample processingfunctionality. Given the benefit of this disclosure, additional objectsand features of the invention, or of certain preferred embodiments ofthe invention, will be apparent to those skilled in the art, that is,those skilled in this area of technology.

SUMMARY

In accordance with a first aspect, an NMR system comprises an NMR probecomprising multiple NMR detection sites. Each of the multiple NMRdetection sites comprises a sample holding void and an associated NMRmicrocoil. The NMR system further comprises a controllable fluid routeroperative to direct fluid sample to the multiple NMR detection sites. Inaccordance with certain preferred embodiments, the multiple NMR sitesare integrated in a probe module further disclosed below. In accordancewith certain preferred embodiments, each of the NMR detection sites isin a capillary-scale fluid channel in the module. In accordance withcertain preferred embodiments, each of the NMR detection sites is in amicro-scale fluid channel in the module. In accordance with certainpreferred embodiments, the controllable fluid router is operative inresponse to an electrical input signal, especially to direct fluidsample to any selected ones of the NMR detection sites. In accordancewith certain preferred embodiments, the NMR system further comprises acontroller unit in communication with the router and operative togenerate the input signal to the router. In certain embodiments the NMRsystem controller unit is operative to receive information from any ofthe multiple NMR detection sites and to generate the input signal to therouter based at least in part on that information. In accordance withcertain preferred embodiments, the NMR system further comprises a dataprocessing unit which may be remote from the probe module or integraltherewith. The data processing unit can provide the aforesaid inputsignal to the controllable router.

In accordance with a second aspect, an NMR probe comprises multiple NMRdetection sites as disclosed above, each comprising a sample holdingvoid and an associated NMR microcoil, and a controllable fluid routeroperative to direct fluid sample to the multiple NMR detection sites.

In accordance with another aspect, an NMR “smart probe” comprisesmultiple NMR detection sites, each comprising a sample holding void andan associated NMR microcoil, a controllable fluid router operative inresponse to an electrical input signal to direct fluid sample to themultiple NMR detection sites, and a controller unit in communicationwith the router and operative to generate the input signal to therouter.

In accordance with another aspect, an NMR probe module is provided,e.g., a module suitable to be interchangeably (i.e., removeably andoptionally reuseable) installed in certain preferred embodiments of theNMR probes disclosed above. Such probe modules comprise at least onefluid inlet port operative to receive a fluid sample, a fluid pathwaycomprising multiple channels in fluid communication with the inlet port,for the transport of fluid sample to be tested, multiple NMR detectionsites, each in fluid communication with at least one of the multiplechannels and each comprising a sample holding void and an associated NMRmicrocoil, and a controllable fluid router operative to direct fluidsample in the module to at least a selected one of the multiplechannels.

The multiple NMR sites optionally can be optimized for different nuclearspecies and/or for 1 or 2 dimensional NMR study, e.g., the sites can beoptimized similarly or differently, using different materials, such asfused silica and PEEK, fused silica and polytetrofluoroethylene and/orother suitable materials known to those skilled in those skilled in theart.

In accordance with another aspect, an NMR probe module comprises atleast one fluid inlet port, operative to receive a fluid sample, a fluidpathway comprising multiple channels in fluid communication with the atleast one fluid inlet port, for the transport of fluid sample to betested, and multiple NMR detection cells, each in fluid communicationwith a corresponding one of the multiple channels. Each of the multipleNMR detection cells comprises an associated NMR microcoil and anenlarged void for holding a fluid sample. In certain preferredembodiments. the NMR probe module further comprises a controllable fluidrouter as disclosed above.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofan illustrative embodiment taken in conjunction with the accompanyingdrawings in which:

FIG. 1 is a schematic block diagram of a preferred embodiment of the NMRsystem disclosed here;

FIG. 2 is a schematic illustration of a preferred embodiment of a NMRprobe and other components of the system of FIG. 1;

FIG. 3 is a schematic view, partially broken away, of a NMR detectionsite of the system of FIG. 1 in a preferred orientation relative to themodule; module

FIG. 4 is an enlarged schematic view, partially broken away, of thesample holding void and associated microcoil of the NMR detection siteof FIG. 3, showing a further enlarged view of same;

FIG. 5 is a perspective view of the enlarged void of a NMR detectioncell in accordance with a preferred embodiment, the NMR microcoil beingbroken away;

FIGS. 6–8 are schematic illustrations of alternative preferredembodiments of NMR probe modules, each showing detection siteorientation, with the top of the page being upward into an NMR magnet;

FIG. 9 is a schematic perspective view fan embodiment of a NMR probe,showing the orientation of a probe module within the probe;

FIG. 10 is a schematic perspective view of an embodiment of a NMR probe,showing in enlarged break-out view the orientation of a probe modulewithin the probe; module

FIG. 11 is a schematic perspective view of an embodiment of a NMR probecomprising operative components in communication with a probe module,showing in enlarged break-out view the orientation of the probe modulewithin the probe;

FIG. 12 is a schematic cross-sectional elevation view of afluid-handling substrate suitable for use as a NMR detection module asdisclosed here;

FIGS. 13A–13D are schematic cross-sectional views of four alternativeconfigurations for fluid channels in a probe module;

FIG. 14 is a schematic view of an NMR detection site in a probe module;and

FIG. 15 is a schematic view of a probe module, showing preferredplacement and orientation of impedance matching elements.

FIG. 16 is an enlarged schematic view, partially broken away, of twosample holding voids in fluidic seies and their associated microcoils ofNMR sample detection sites of the present invention;

FIG. 17 is a perspective view, partially broken away, of the void ofeach of a pair of NMR detection cells in accordance with a preferredembodiment;

DETAILED DESCRIPTION

It will be recognized by those skilled in the art that numerousdifferent embodiments of the systems, probes and modules disclosed herecan be produced and used for various different applications. Incapillary-based embodiments analyte sample fluid has a fluid flow ratetypically less than about 5 μL/minute. In substrate-based, i.e.,micro-scale, embodiments the analyte sample fluid has a fluid flow ratetypically less than 1 μL/minute. In certain preferred embodiments, aminiaturized analysis system is employed for liquid phase sampleanalysis. Such embodiments, referred to in some instances here and inthe appended claims as substrate-based, employ a microfabricated supportbody or manifold in the form of a cylinder, chip, laminated planarsubstrate or the like, insertable, e.g., removeably insertable, such asa set of interchangeable modules or the like. Typically in suchembodiments the module has one or more straight or branchedmicrofabricated microchannels and the probe has an inlet port forfeeding fluid from an external source into the manifold. Multiple NMRdetection sites each comprising an NMR RF microcoil in the module are influid communication with the inlet via one or more of themicrofabricated microchannels. As used here, the terms “micro-scale” and“microfluidic” means the manifold operates effectively on micro-scalefluid samples, typically having volumes less than about 1 uL (i.e., 1microliter), e.g., about 0.1 microliter to 1.0 microliter, and fluidflow rates less than about 1 uL/min, for example 100 nanoliters/min. Theterm “microscale” also refers to flow passages or channels and otherstructural elements of a substrate, e.g., a multi-layer laminatedsubstrate. For example, 1 or more microchannels of a module substratepreferably have a cross-sectional dimension (diameter, width or height)between 500 microns and 100 nanometers. Thus, at the small end of thatrange, the microchannel has cross-sectional area of about 0.01 squaremicrons. Such microchannels within a laminated substrate of the module,and chambers and other structures within the laminated substrate, whenviewed in cross-section, may be triangular, ellipsoidal, square,rectangular, circular or any other shape, with at least one andpreferably all of the cross-sectional dimensions transverse to the pathof fluid flow is microscale. It should be recognized, that one or morelayers of a laminated substrate may in certain embodiments haveoperative features, such as fluid channels, reaction chambers or zones,accumulation sites etc. that are larger than microscale. The modulesdisclosed here provide effective microcoil NMR devices and systems withgood speed of analysis, decreased sample and solvent consumption,increased detection efficiency, and in certain embodiments disposablefluid-handling devices.

The multiple NMR detection sites preferably are used in systems furthercomprising one or more analyte extraction chambers. Thus, analyte fluidsample may be produced in an analyte extraction chamber and fed,directly or indirectly (i.e., with or without intervening processing orstorage) to the multiple NMR detection sites. The detection sites may beused in parallel, in series or as alternatives to each other depending,e.g., on characteristics of the analyte sample. Optionally suchcharacteristics may be determined by an operative component of thesystem, e.g., an operative component integrated on-board an embodimentof the probe modules disclosed here. The term “analyte extractionchamber” means a column or other operative zone or site or the like forfocusing or concentrating analyte from a sample fluid into an analytesample fluid, typically involving a many-fold reduction in fluid volume.The analyte extraction chamber may be in a separate or stand-alonedevice, e.g., an LC column, with fluid communication to the NMR probevia any suitable conduit, e.g., a fluid delivery tube controlled by anautosampler. The analyte extraction chamber preferably is acapillary-based analyte extraction chamber integrated into the NMRprobe, e.g., as an operation site along a fluid channel extending withinthe probe to the NMR detection site or is integrated on-board asubstrate-based module. Preferably, the analyte extraction chamber isoperative to perform liquid chromatography, capillary electrophoresis,dynamic field gradient focusing, electric field gradient focusing or thelike. In the case of an LC column in the form of a capillary-basedanalyte extraction chamber operative to perform solid phase extraction(SPE) or integrated into a substrate-based NMR probe (in some instancesreferred to here as an on-board LC chamber or on-board LC device), ananalyte peak will be stepped off the column or other analyte extractionchamber, i.e., released into the NMR probe fluid channel, when therelative proportion of analyte solvent (e.g., an organic solvent) in theanalyte sample fluid reaches a sufficient concentration.]

Referring now to the drawings, FIG. 1 is a schematic representative ofan electrofluidic system in accordance with the present disclosure. Amultiplicity of sample management modules are in operative electricaland fluidic communication with a multiplicity of primary stagedetectors, a peak management module, and a multiplicity of ancillarystage detectors. Sample introduction can be from a variety ofintroduction means well known to those skilled in the art, and mayinclude autosamplers with or without additional means of solid phaseextraction. In general, the flow of information and fluid transportdepicted in FIG. 1 can proceed in either direction. For example, withthe appropriate plumbing as understood by those skilled in the art, astorage loop used for sample introduction can be reused for samplestorage, e.g. as a fraction collector at the end of the experiment.Furthermore, the figure should be considered sufficiently general as torepresent the combination of any number of individual components, forexample, the case where a single sample management module is used with amultiplicity of ancillary stage detectors. The sample managementplatforms are sufficiently sophisticated to be in operative electricaland fluidic communication with each other. One embodiment of thisconfiguration is where each of the detection stages are NMR microcoildetectors. A preferred embodiment is where all detectors are integratedinto the probe manifold but are not limited to NMR, e.g. UV, IR, andother NMR-compatible (predominantly non-magnetic) means of detection.The peak management module can include sample storage and routingcapabilities, but can also include a means of sample management, e.g.solid phase extraction. In a most preferred embodiment, the componentsshown are predominantly integrated into a probe module, such as those ofFIGS. 3–8, with intelligent control of the overall processes beingdirected at least in part by electrical and fluidic processing elementsin (i.e., on-board) the probe, e.g. microprocessors in operativecommunication with the detectors and fluidic management components shownin the drawings.

FIG. 2 illustrates a preferred embodiment of the system of FIG. 1,including a means of sample management (Waters CapLC and Sparcautosampler) external to the NMR probe, a primary stage detector (Watersphotodiode array) external to the NMR probe, and a capillary-based NMRdetection probe in operative electrical and fluidic communication withthe sample management system and with the NMR spectrometer for effectivecontrol of analysis through a computer external to the probe. In analternative preferred embodiment, some or all of these components wouldreside in (i.e., on-board) the probe, or even be provided as micro-scalecomponents integrated on-board a substrate-based module, therebyimproving efficiency, electrical and fluidic integrity, and promotingintegration and complexity.

FIGS. 3–8 show a key aspects of the NMR probe, i.e., exemplary NMRdetection modules suitable for capillary-scale embodiments. The NMRdetection modules would preferably contain a multiplicity of primaryand/or ancillary NMR detectors, and would be in operative communicationwith the other elements of the system of FIG. 1. FIG. 3 shows a singledetection site in a module, the detection site being defined here as acombination of a capillary having an enlarged region and a microcoil NMRdetector associated therewith. FIG. 4 shows a cutaway view of anotherdetection module embodiment, illustrating the microcoil wrapped on acapillary. FIG. 5 shows an enlarged region of fluidic pathway (innerdiameter of the capillary). FIG. 6–8 show alternative preferredembodiments of geometrical positioning of multiple NMR detection sitesor cells in a single detection module. Although five detection cells areillustrated, the concept presented herein applies generally to anynumber of detection sites in a module. Also, it should be understoodthat alternative embodiments of the probes and systems disclosed heremay employ multiple modules.

FIG. 9–11 show representative embodiments of the NMR probes disclosedhere. FIG. 9 shows a typical probe (wire frame mesh) and illustrates theorientation/position of the detection module relative to a module base.The module would be inserted upwardly (as viewed in the drawing) into anNMR magnet. FIG. 10 shows a module similar to FIG. 9 but having multipledetection sites or cells. FIG. 11 shows another similar probe, buthaving multiple operative components integrated into the probe,specifically, plug-in modules (PIMs). The PIMs can be in operatively inelectrical communication and/or operatively in fluidic communicationwith one or more detection sites, and may contain fluidic (e.g., samplemanagement, e.g. LC, CE, DFGF) processors or electrical (e.g.electronic, hardware, software, e.g. digital computer) processors.

Referring to FIG. 12, a laminated substrate of a substrate-based moduleis seen to comprise, a first plastic piece 10 and a second plastic piece11 welded together by selective IR irradiation of either the plasticpieces or by irradiation of an optional EM absorbing substance 12. Thesubstrate contains a channel 13 formed by welding of the two plasticpieces together. Optionally contained within the channel 13 is anenvironmentally sensitive element 14. The substrate may also containother channels formed from welding the plastic pieces together. A secondchannel 15 is in close and continuous contact with an embeddedmicrodevice 16. A port 17 provides communication from the channel to thetop or bottom planar surface of the substrate. Additionally, an externaldevice may be connected to the fluid-handling substrate through theport. An optional gasket 18 may be used to enhance the fluid-tight sealaround the channel. An optional EM absorbing layer 19 may be placedanywhere along the surface of the substrate. FIG. 13 showscross-sectional views of four alternative configurations for fluidchannels in the probe module. Such channels may be formed, for example,by welding module layers e.g. plastic pieces together. Possibleconfigurations include, but are not limited to, semi-circular 21,rectangular 22, rhomboid 23, and serpentine 24. The channelconfigurations are limited only by the thickness of the materialsforming the fluid-handling substrate.

In accordance with one embodiment, a NMR probe module comprises asubstrate defining at least one fluid inlet port, and a fluid pathwaycomprising multiple channels in fluid communication with the at leastone fluid inlet port, for the transport of fluid sample to be tested;multiple NMR detection sites each in fluid communication with at leastone of the multiple channels, each comprising a sample holding void, andan associated RF microcoil; and a controllable fluid router operative inresponse to an electrical input signal to direct fluid sample in themodule to at least a selected one of the multiple channels correspondingto the input signal.

The module may be any size and shape suitable to the intendedapplication. In a preferred embodiment the module may be placed in ahousing that forms a housing of the probe. In certain embodiments theprobe is tubular or finger shaped and/or boxed-shaped to match therepresentative forms of NMR probes, with for example, a diameter ofabout one inch and a height of about 20–30 inches. In certainembodiments the module is generally planer. The term “generally planer”means card or cartridge-like, optionally being curvo-planar or otherwiseirregular, but otherwise typically being rectangular orright-cylindrical, and having a thickness less than about a third,preferably less then one quarter, more preferably less than one fifthe.g. about one sixth or less, the largest dimension of major (i.e.largest) surface of the substrate. Other embodiments will be apparentgiven the benefit of this disclosure.

The microfluidic nature of the NMR probe modules disclosed here providessignificant commercial advantage over conventional (larger scale, e.g.larger than capillary) fluidic NMR systems. Less sample fluid isrequired, which in certain applications can present significant costreductions, both in reducing product usage (for example, if the testsample is taken from a product stream) and in reducing the waste streamdisposal volume. In addition, the microfluidic substrate assemblies can,in accordance with preferred embodiments, be produced employing MEMS andother known techniques suitable for cost effective manufacture ofminiature high precision devices.

The module may be made of any number of materials. Examples includemetals, plastics, and silica. In a preferred embodiment the manifoldcomprises a substrate formed at least in part of polyetheretherketone(PEEK). PEEK is a high temperature resistant thermoplastic. PEEK hassuperior chemical resistance allowing for its use in harsh chemicalenvironments, and it retains its flexural and tensile properties at veryhigh temperatures. Additionally, glass and carbon fibers may be added toPEEK to enhance its mechanical and thermal properties. In anotherembodiment the module is a multi-layered laminate. Other embodimentswill be apparent to one skilled in the art given the benefit of thisdisclosure.

In accordance with other embodiments the module further comprises afluid outlet port in fluid communication with the fluid pathway. Inother embodiments the module further comprises a fluid reservoir influid communication with the fluid pathway.

The fluid inlet port is where a fluid sample to be tested is introducedto the module. The fluid inlet port is typically sized to accommodatethe amount of sample being tested. The inlet port may have any number ofshapes. Examples include circular, square, trapezoidal, and polygonal.The inlet port may or may not also include additional filtering for thefluid sample to be tested. In certain embodiments there may be multipleinlet ports. Further embodiments will be apparent to one skilled in theart given this disclosure.

The fluid pathway transports the fluid sample to be tested though-outthe module. In a preferred embodiment where the probe module ismicrofluidic the scale the multiple channels (sometimes referred to asmicrochannels) may be capillary in scale. The orientation of thechannels may be any number of configurations. Examples include parallel,intersecting, overlapping, spiral, serpentine, and circular. The crosssection of the channels may have any number of shapes as well. Examplesinclude circular, square, trapezoidal, and polygonal. Furtherembodiments of size, shape, and orientation will be apparent to oneskilled in the art given the benefit of this disclosure.

The multiple NMR sites are provided to allow for increased functionalityand/or throughput. With multiple NMR sites the user is able to performmultiple NMR tests simultaneously which increased the rate in whichresults may be obtained. Furthermore the NMR detection sites may beoptimized for different types of testing allowing a single probe to beused for a number of tests. In some embodiments each NMR site maybe influid communication with multiple channels of the fluid pathway. Inaccordance with preferred embodiments, the NMR detection sites furthercomprise matching capacitors and tuning capacitors, fluid connectors anddata transmission means such as signal carrying leads or the like. Anexample of an NMR detection site can be seen in FIG. 14. Otherembodiments will be apparent to one skilled in the art given the benefitof this disclosure.

The multiple NMR detection sites may be optimized using differentmaterials. In a preferred embodiment the multiple NMR detection sitesare made of fused silica and PEEK. In another preferred embodiment themultiple NMR detection sites are made of fused silica and Teflon. Otherembodiments will be apparent to one skilled in the art given the benefitof this disclosure.

In a preferred embodiment the sample holding void is cylindrical inshape but it may also have any number other shapes, most notablyspherical. Examples of cross-sectional configurations include round,rectangular, triangular, etc. In a preferred embodiment the sampleholding void is 5 um to 500 um, more preferably 25 um to 50 um. In apreferred embodiment the microcoil surrounds around a portion of thevoid. Preferably the microcoil is made of copper but may be made of anynumber of other conductive or super-conductive materials depending onthe desired properties. The microcoil typically is 250 um to 1 mm inaxial direction. In preferred embodiments the microcoil may be helical,solenoidal or spiral and in other preferred embodiments the microcoilmay be planar. Other embodiments of coil geometry will be apparent toone skilled in the art given the benefit of this disclosure.

In the NMR probe module configuration disclosed here, each of the NMRdetection sites is separate and therefore, optionally, can hold uniquesamples for testing. In this regard, the probe modules integrate amultiplicity of detectors with greater functionality enhancement thanwould be achieved by wrapping a multiplicity of detection coils around asingle sample. The microcoil, void, and sample are magnetically matched,and the NMR detection sites in accordance with preferred embodiments areoperative to obtain high resolution NMR spectra. The microcoil ispositioned to within 1 mm, more preferably to within less than 100 um ofthe sample boundary. The incorporation of multiple microcoils andmultiple corresponding voids complicates magnetic matching, but will bewithin the ability of those skilled in the art given the benefit of thisdisclosure and applying known principles. Preferably, at least onedetection coil is optimized for high resolution proton detection,whereas other coils may be optimized for heteronuclear ormulti-dimensional homonuclear experiments. In two-dimensionalexperiments such as correlation spectroscopy (COSY) or total correlationspectroscopy (TOCSY), and in heteronuclear experiments the digitalresolution in the f1 dimension is relatively coarse (typically 128–256data points). The number of data points in the f2 dimension (typically512) is, therefore, considerably reduced compared to one-dimensionalexperiments, and the acquisition time is similarly reduced (˜200 ms at250 MHz, and shorter at higher operating frequencies). Consequently, theresolution requirements of coils intended for 2-D acquisition isconsiderably lower due to the larger spectral linewidths (typically 2–4Hz) in 2-D experiments. For example, in an embodiment with 2 detectionsites, a primary coil can be optimized for resolution while a secondarycoil can be optimized for heteronuclear acquisition.

The functionality of the NMR probe module is dependent, in part, uponthe reception of separate signals from the individual coils (andimpedance matching networks) that comprise the NMR detection site.Regardless of the form of signal acquisition (independent acquisitionusing multiple receivers, or time-multiplexed acquisition using RFswitches), quasi-simultaneous acquisition demands a high degree ofisolation between the microcoils and matching circuits at each nuclearfrequency of interest. The coils can be positioned with spatialseparation and orthogonal geometric orientation to reduce coupling.Similarly, the impedance matching circuits can be shielded andpositioned in the probe in such a manner to reduce coupling.

In accordance with another preferred embodiment, the timing constraintsof a multiplexed system for NMR applications (where timing is acritically important parameter) can be addressed by the use of amultiple transceiver NMR spectrometers.

This advance of high potential significance is made technically possibledue to the relative size of the RF microcoil (typically 1 mm diameter)when compared to the overall size of the (shimmed) region of fieldhomogeneity in the NMR magnet. Magnets are typically designed forconventional RF coils and samples as large as 1 cm in diameter andextending (vertically) several centimeters in length. By reducing thesize of the RF coil by as much as 10-fold, the possibility ofincorporating several coils into one magnet becomes feasible. Theorder-of-magnitude advantages in mass sensitivity (when compared toconventional NMR probes) achieved using a NMR probe module disclosedhere, and the chromatographic resolution that can be achieved andmaintained (e.g. when employing liquid chromatography for samplepreparation) at the capillary-scale have been extensively documented.

Important advantages obtained by employing preferred embodiments of theNMR probe module disclosed here include high throughput due toparallelism of detectors, while retaining the inherent gains in masssensitivity and chromatography afforded at the capillary size scale andusing microcoils, individual optimization of RF coil sensitivity todifferent nuclei (1H, 1H{15N}, 1H{13C}, etc.), e.g. where peaks arerouted sequentially through a series of optimized detectors (therebysaving the money and time associated with use of multiple probes whilestill preserving uncompromised sensitivity, time optimization of dataacquisition, e.g. by using a dedicated coil for rapid 1-D analysis whilereserving another for 2-D, longer acquisition NMR experiments, and thecreation of a platform for eventual integration o intelligent samplemanipulation and NMR analysis, whereby the directed routing of analytepeaks can be accomplished based upon the content of the peak (using anappropriate sample management technology).

Li et al. (Li 1999 Anal. Chem. 71 4815–4820) describes a 4-coil assemblyillustrative of certain aspects of the present disclosure. Thesolenoidal microcoils (diameter=360 :m, length approx. 1 mm) are mountedon horizontal (transverse to B₀) capillaries with a 90 degree rotation(x, y) and 5 mm vertical spacing between adjacent coils. Additionaldetails of basic construction are known generally, as shown in the Li etal reference mentioned above and incorporated herein by reference forall purposes. The NMR probe modules disclosed here differ from suchearlier devices in having multiple detectors, each having a detectionsite, i.e., as described above, void in the capillary microchannel toreceive a test sample, and having an NMR microchannel aligned therewith.

It is generally preferred to immerse the diamagnetic wire of themicrocoil in a diamagnetic matching medium with magnetic susceptibilityequal to that of the wire. Suitable software is commercially availablefor use in determining determine inter-coil spacing, coil orientations,and other geometrical tradeoffs for optimum resolution, for example,Maxwell 3D (Ansoft Corporation, Pittsburgh, Pa.). Preferably, greaterthan 30 dB RF isolation is achieved between coils. Coil-to-coil couplingwould result in cross-talk and interference in the received signals. RFcoupling is typically minimized by geometrically positioning electricalcomponents so that the orientation of the magnetic and/or electricfields in adjacent components are orthogonal. Adjacent components arealso placed as far apart as is practical to maintain functionality butminimize coupling.

Shielding (via insertion of a conducting plane) can be used effectivelyfor elimination of electric field coupling, albeit at the expense of anincreased effective resistance (and noise) due to induced eddy currentsin the shield. The increased resistance typically manifests as a loweredquality factor (Q) for the resonant circuit. Magnetic field coupling ismore difficult to restrict, as the lines of magnetic flux close uponthemselves. In non-magnetic environments, high permeability (mu) metalsare employed, but these materials are precluded in NMR due to theireffect on field homogeneity. Preferably a combination of orientation andspacing for minimization of coupling are used. Preferably the matchingnetworks are spatially separated in the probe. FIG. 15 illustrates anexemplary probe layout demonstrating spatial and electrical separationof RF impedance matching circuit components, e.g. to achieve isolationin 1H/2H circuits. The NRM probe modules disclosed here preferablyemploy a multi-level geometry, where adjacent matching networks can beshielded using copper sheets or the like, that isolate the electricfields of one matching circuit on one level of the probe from those ofanother on a different level of the probe. Circuit modeling, e.g., usinga commercial RF package such as Linc2 available from AppliedComputational Sciences (Escondido, Calif.) can be employed to help guidethe design. A variety of options exist for further probe modification,including the incorporation of additional shielding to partitionadjacent circuits on the same level.

The NMR probe module may have each of the multiple NMR detection sitesoptimized differently with any of the combinations discussed above orany other that may be apparent to one skilled in the art given thebenefit of this disclosure. FIG. 16 shows two sample holding voids influidic series. Specifically, in the embodiment of FIG. 16, a firstsample detection site 40 and a second sample detection site 41 are seento be in fluidic series with each other. Each of the fluidic series NMRdetection sites 40 and 41 is seen to have associated with it acorresponding microcoil. FIG. 17 shows a pair of NMR detection cells 50,51 in accordance with a preferred embodiment. The cells 50, 51 are influidic series with each other along a capillary tube and are shownwithout associated coils.

The microchannels and associated NMR microcoils can be formed in amodule, preferably a multi-layer substrate, such as a laminatedmulti-layer substrate, e.g., a selectively welded multi-layer substrateas disclosed in copending U.S. patent application Ser. No. 60/239,010filed on Oct. 6, 2000, relevant portions of the disclosure of which areset forth immediately below in the fifteen paragraphs section “LaminatedMultilayer Substrates”. Microlithographic microcoils can be employed insuch laminate substrates, such as those disclosed in the above-mentionedU.S. Pat. No. 5,684,401, the entire disclosure of which is incorporatedherein by reference for all purposes. Alternatively, or in addition, oneor more of the multiple NMR detector sites formed in the probe can beformed in a finger or peninsula-type extension of the substrate, and themicrocoil can be formed as a separate 3-dimensional structure fittedover such substrate projection. It will be within the ability of thoseskilled in the art, that is, those skilled in this area of technology,given the benefit of this disclosure, to employ alternative suitablefabrication techniques for production of the multi-microcoil NMRdetection probes disclosed here.

Laminated Multilayer Substrates: The fluid handling devices disclosedhere may be conveniently constructed by forming the flow passages andseparator in the surface of a suitable substrate layer, such as a layerof flexible or rigid plastic or other material, and then laminating theadjacent layer to the first layer. Micromachining technology is known,which is suitable for the manufacture of at least certain embodiments orcertain portions of the microfluidic substrate assemblies disclosedhere, having elements with minute dimensions, ranging from tens ofmicrons to nanometers. A portion of one or more substrate microchannelsmay be formed in one or more of the substrate layers, such that thecomplete channel is only formed when the layers are joined together. Thepieces are joined together in a fluid-tight manner to seal the channel,e.g., to form a closed (i.e., fluid-tight) periphery for the channel,such as for the transport of fluids. Closing or welding the piecestogether to form and seal the channels can be accomplished in a numberof known ways. One such method involves assembling, i.e., positioningthe pieces together and heating the assembly to the melting point, or atleast the softening point, of one or both of the pieces (or all of thepieces where more than two pieces are assembled together). Adhesivemethods also are known for assembling the miniaturized fluid-handlingsubstrates.

Microfluidic substrate assemblies disclosed here, having a multi-layerlaminated substrate, can be designed and fabricated in large quantitiesusing known micromachining methods. Such methods include film depositionprocesses, such as spin coating and chemical vapor deposition, lasermachining or photolithographic techniques, e.g. UV or X-ray processes,etching methods which may be performed by either wet chemical processesor plasma processes LIGA processing and plastic molding. See, forexample Manz et al., Trends in Analytical Chemistry 10:144–149 (1991).More generally, the design and construction of microfluidic substrateassemblies disclosed here can commence with computer aided design of thedevice. Optionally, rapid prototyping of the device can be performed,e.g., using laser machining and micro-milling to quickly produce smallquantities. Production quantities are advantageously produced using LIGAand electroforming techniques to produce a master, such as a nickelmetal master. The master can be used in the production of relativelylarge numbers of units through injection molding and embossingtechniques. Finished devices typically will require additionalproduction steps, such as coating, packing and filling steps inaccordance with known manufacturing techniques.

In accordance with certain preferred embodiments selective welding isaccomplished by IR radiation. The substrate formed in this way has oneor more internal fluid channels, and may be essentially planar orblock-like in configuration. Also, the substrate assembly may be weldedor otherwise joined to other pieces or components, such as to form acartridge to be inserted into a corresponding socket or port to formfluid-tight seals with fluid lines communicating with a process linecarrying fluid to be analyzed or detected or the like. The selectivewelding of substrate pieces together, e.g., two or more planar plasticpieces to be stacked together and selectively welded to form sealsestablishing fluid-tight channels within the resulting body, utilizes IRradiation, laser or the like, on the areas of the plastic pieces to bejoined. This process is usually done by positioning two substrate piecesin direct and continuous contact with one another and subsequentlyexposing the pieces to radiation.

Taking a preferred embodiment of plastic substrate layers to illustratethis aspect, one of the plastic or other material pieces may betransparent to the radiation while the other is opaque to radiation.Alternatively a radiation absorbing material can be dispersed within oneof the plastic pieces, either selectively in the area to be welded orthroughout the body of the material forming the piece. Alternatively aradiation absorbing material can be coated on the surface of one or bothof the pieces, either selectively in the area to be welded or all over.Where selective absorption is not established, the use of focused ormasked radiation or the like can be used to accomplish the selectivewelding. It should be recognized that selective welding of an interfacebetween two substrate pieces assembled together may in some embodimentsinclude irradiation and welding of the entire interface. Thedisadvantages discussed above of thermal welding are still avoided,since it is not necessary to heat the substrate assembly in its entiretyto the melting or welding temperature. It is the joint region orinterface of the two plastic pieces that is exposed to radiation,forming the selective weld. Again using plastic substrate pieces toillustrate this aspect, the radiation from a laser beam or otherradiation source can pass through a transparent plastic piece and intoan opaque plastic piece. Melting of the opaque plastic piece results asthe incident radiation is absorbed by the opaque plastic piece. Removalof the radiation results in cooling and formation of a weld between thetwo plastic pieces.

In published PCT application No. WO 00/20157, a method of forming a weldbetween two workpieces is taught, one of the pieces being opaque and theother being transparent to radiation. It also teaches a method ofproviding a radiation absorbing material at the joint region of the twoworkpieces, where both plastic pieces are transparent, in order to forma weld between them. Infrared radiation (IR) bonding has been used tojoin plastic articles, as in U.S. Pat. No. 6,054,072, the entiredisclosure of which is incorporated herein by reference. The use of suchtechniques in the methods disclosed here and the advantages in themethods disclosed here will be apparent to those skilled in the artgiven the benefit of this disclosure.

The plastic components of the fluid-handling substrate described hereinare preferably made of, but not limited to, materials selected from thegroup consisting of polysulphone, PEEK, polyfluoroethylene (PFE),polycarbonate, ceramic, Teflon, stainless steel, polydimethylsiloxane(PDMS), pyrex, soda glass, CVD diamond, PZT, silicon nitride, silicondioxide, silicon, polysilicon, Au, Ag, Pt, ITO, and Al. PEEK is apreferred material for the plastic pieces and components to be made frombecause it is chemically inert and is insoluble in all common solvents.It is also resistant to attack by a wide range of organic and inorganicchemicals. PEEK has excellent flexural, impact, and tensilecharacteristics. PEEK is especially advantageous because it has a lowglass transition temperature (Tg) and will weld at a temperature thatwill not lead to the distortion, warping, or destruction ofenvironmentally sensitive elements contained within the plastic pieces.Additionally, PEEK allows for visualization during the welding processand for visual inspection of the seals created by the welding process.

The substrate contains a channel formed by welding of the two plasticpieces together. The cavities or chambers within the plastic pieces thatform the channels (after the plastic pieces are welded together) can beformed into the plastic pieces using any method known in the artincluding, but not limited to, UV-embossing, heat-embossing, laserablation, injection molding, CNC micro-milling, silicon micro-machining,focused ion beam machining, wet etching, and dry etching. The channelscan be of a large variety of configurations, such as, a straight,serpentine, spiral, or any path desired. Also, a wide variety of channelgeometries including, but not limited to, semi-circular, rectangular,rhomboid, and serpentine can be formed and are limited only by thethickness of the materials forming the fluid-handling substrate. Thechannels may be one dimensional or multidimensional (two-dimensionalor-three dimensional). As used herein, the term one dimensional channelmeans a channel that runs along a single axis aligned with the plane ofthe substrate. The term multidimensional channel, as used herein, meansa channel that runs along two or more axes, perpendicular to each other,in the plane of the substrate. The resulting dimensional aspects andarchitecture of the channels are especially sensitive to hightemperature conditions because they can warp to the point at which theywould no longer be functional or maintain the desired shape.

As disclosed above, in certain preferred embodiments a microchannel isformed in the multi-layer laminated substrate at the interface of twolayers. It is an advantageous aspect of these preferred embodiments thatthe layers are effectively welded or otherwise joined to form afluid-tight seal along the periphery of the channel. A fluid-tight sealis a seal in which the channels do not leak fluid, that is,substantially no fluid can enter or exit the channels through the sealedperiphery, but rather only through fluid communication ports provided.It will be understood from this disclosure, that such fluid ports can bepositioned at any convenient location in the surface of the substrate,taking into account the need to provide fluid channels within thesubstrate to the port. The port may be located on either a major surfaceor on any side surface, hereafter referred to as a minor surface, of thesubstrate. An embedded microdevice can be contained within a secondchannel or chamber. Both fluid channels can be formed by and at theinterface of the two substrate layers. The port and microchannel can beany suitable configuration, such as, straight, serpentine, spiral etc.Also, a wide variety of port geometries including, but not limited to,semi-circular, rectangular, and rhomboid can be formed and are limitedonly by the thickness of the materials forming the fluid-handlingsubstrate.

Additionally, as discussed below, the port may in certain preferredembodiments be employed as a docking site for a component-on-board,i.e., an external device mounted to the substrate for increasedfunctionality, more specifically, a mounted component that will be influid communication with a microchannel in the substrate. A gasket 18may be used to form or enhance a fluid-tight seal between a mountedcomponent-on-board and the surface of the substrate. A gasket, asreferred to herein, may be an “O” ring carried by the mounted componentor by suitable structure of the substrate. In certain preferredembodiments, curable gaskets are employed at the mounting site of anon-board component. Such gaskets can be usefully formed of radiationabsorbing materials, such as plastics or metals, and preferably have alower Tg than the adjacent materials of the substrate and on-boardcomponent. After the component is positioned on the substrate the gasketat the joint between them is subjected to actinic or curing radiation.Also, suitable gaskets can be microformed on or in the surface of thelaminated substrate and/or the surface of the component to be mounted. Agasket can also be employed that covers the entire contact surface ofthe substrate and the component.

Assembly of the fluid-handling substrate occurs as the substrate piecesare welded together and the channels are preferably sealed usingselective EM welding techniques, such as selective IR welding.Selectively welded, as used herein, refers to a weld that produces afluid-tight seal surrounding the channels in the plastic pieces orcomponents of the fluid-handling substrate. The selective welding ispreferably done substantially in the area immediately surrounding thechannel the weld is intended to seal. However, this does not exclude anywelding location that may create a fluid-tight seal. The most preferablewelding methods include, but are not limited to, IR dosage (pulsed,continuous, intensity, frequency/bandwidth), IR delivery (spot, flood),thermal conditions (workpiece, platen(s), pick tools), ultrasonicagitation, or pressure. The chambers or cavities responsible for formingthe channel after the pieces have been welded together can be machinedinto the plastic pieces using any method known to those skilled in theart. The welding of the plastic pieces is done by first aligning themajor surface of the first plastic piece and the major surface of thesecond plastic piece using mechanical means. Next, an EM beam is appliedthrough the surface of the transmissive plastic piece. The EM opaquefirst plastic piece will absorb the energy of the EM, and heat will begenerated causing the surface of the plastic pieces to melt or soften.The melted surface will cool, and the plastic pieces will then be weldedforming a channel with a fluid-tight seal.

In accordance with preferred embodiments, the plastic pieces and gasketsare preferably made of PEEK as this material provides for thepossibility of visual or optical inspection of the weld and resultantfluid-tight seal. Additionally, other properties of PEEK make its usedesirable. PEEK has superior chemical resistance allowing for its use inharsh chemical environments. PEEK retains its flexural and tensileproperties at very high temperatures. Additionally, glass and carbonfibers may be added to PEEK to enhance its mechanical and thermalproperties. One advantage of using PEEK in the assembly of afluid-handling substrate, as discussed above, is that the selective IRwelding process may be visually or optically monitored, as PEEK is aclear and colorless material. Therefore, the fluid-tight seals that arecreated, using the selective IR welding process, may be inspected priorto further assembly or distribution of the fluid-handling substrate. Ifupon visual or optical inspection it is determined that the seal is nota fluid-tight seal, additional selective welding can be performed priorto testing of the fluid-handling substrate, thus the ciuality of theassembled fluid-handling substrate is much higher than those producedusing current methods.

In accordance with certain preferred embodiments, joining of plasticpieces and sealing of channels can be accomplished with a focusable EMbeam, such as a laser. As used herein, the term focusable EM beam refersto any light source where the size of the light incident on the surfaceis very small when compared to the overall size of the surface, whereasan EM beam refers to any light source that may illuminate a significantportion or all of a surface. An advantage of using a focusable beamincludes direction of the radiation away from any areas that might bedamaged from the radiation, such as those areas containing anenvironmentally sensitive component, for example. A fluid-tight seal maythen be created without risking damage to the environmentally sensitivecomponent. The focusable beam may also be coupled with the use of a dyefor time scheduled selective welding. As used herein, time scheduledselective welding refers to using different dyes between each layer, orcoated on or contained within different portions, of the fluid-handlingsubstrate. Two or more dyes can be used to ensure only those areascontaining the appropriate dye are welded together. In this illustrativeexample, two dyes, Epolight 5010 and Epolight 6084, both from Epolin,Inc. (Newark, N.J.). are coated independently on different portions ofthe fluid-handling substrate to be assembled. Epolight 5010 has amaximum liaht absorption at about 450 nm while Epolight 2057 has amaximum absorption at about 1064 nm. Therefore, radiation having awavelength of 1064 nm. such as an infrared laser, would only be absorbedby the Epolight 2057, and only the areas of the fluid-handling substratecontaining the Epolight 2057 would be welded together. A differentradiation source having a wavelength of about 450 nm, such as an argonlaser, would be reciuired to weld any areas containing the Epolight5010. One skilled in the art would recognize that a focusable EM beamcould be used in combination with multiple dyes for time selectivewelding and increased protection of environmentally sensitivecomponents. Additionally, a tunable dye laser could be used to providerapid switching of the incident wavelength and thus providing more rapidmeans for the selective welding process and completion of assembly ofthe fluid-handling substrate. Additional materials suitable for use asIR absorbing materials include high temperature dyes, also availablefrom Epolin, Inc.: Epolight 3079, Epolight 4049, Epolight 3036, Epolight4129, Epolight 3138, and Epolight 3079.

In the event that the pieces of the substrates are both EM transmissive,the pieces may be coated with a substance that is EM opaque. The EMabsorbing substance may be any substance capable of absorbing theincident radiation. Preferred EM absorbing substances include, but arenot limited to, dyes and pigments, for example. Epolight 5010, Epolight5532, Epolight 6034, and Epolight 1125, all from Epolight. Inc.,(Newark, N.J.). FIG. 4 shows a possible configuration for assembly ofthe fluid-handling substrate where both pieces of the substrate are EMtransmissive. When joining plastic pieces that are all EM transmissive,it is necessary to either coat the surface of one or more of the plasticpieces with an EM absorbing substance to form an EM absorbing layer,incorporate an EM absorbing substance into at least one of the plasticpieces. EM interfaces, composed of any EM absorbing substance such asdyes or dye-containing substances, can be created by contrastingadministration regimes including, but not limited to spin-coating,micro-dispensing, and micro-contact transfer printing. A coating of anEM substance is first applied to a major surface of the first or secondplastic piece or both. The plastic pieces are then aligned usingmechanical means. An EM beam is applied through the surface of one ofthe transmissive plastic pieces so that radiation is incident on thecoating. Heating and subseciuent cooling of the EM coating results inwelding of the two plastic pieces together, and formation of a channelwith a fluid-tight seal. A gasket may be used to further enhance theeffectiveness of the fluid tight seal.

An aspect of preferred embodiments is a method for assembly of afluid-handling substrate comprising environmentally sensitivecomponents, as discussed above. For additional protection of theenvironmentally sensitive components, the stacked plastic pieces can bemasked with an EM absorbing substance and only the unmasked portions areexposed to the EM radiation and, therefore, only those locations areheated to seal the plastic pieces. The use of blocking materials confersspatially and/or temporarily selective protection/deprotection of theenvironmentally sensitive components in the channels from the EM. Thesemethods prevent the environmentally sensitive element from becomingheated and subsequently destroyed by the heat from the sealing process.A gasket placed around the resulting channel acts to increase theeffectiveness of the fluid-tight seal. If a focusable EM beam is used,as discussed above, the aligned plastic pieces can be moved in relationto the EM beam to facilitate joining of the correct positions on theplastic pieces. Alternatively, the beam can be moved in relation to thealigned plastic pieces. These two methods allow for greater control overthe portions of the fluid-handling substrates that are irradiated,heated, and sealed.

The radiation necessary to weld the plastic pieces together may beadministered using several different methodologies including, but notlimited to, fiber delivery, controlled spot size and controlled spotintensity, seam forming, and large area rastering. Preferred joiningmethodologies for the plastic pieces and/or components include IRdosage, IR delivery, thermal conditions, ultrasonic agitation, andpressure. The EM radiation source may be any type of EM source,including commercially available lamps or lasers. The EM radiation mostpreferable is infrared radiation (IR) with the JR source preferablybeing infrared lasers or infrared heat bulbs having tungsten filamentsand integral parabolic reflectors. The EM source may optionally includelenses that vary the focal point of the beam. The EM source is generallypositioned and tuned to project the EM beam that passes through the lensand onto the plastic pieces mating surfaces. It will however, berealized that any EM source may be used providing a suitable EMabsorbing material is available, and, if appropriate, one plastic pieceis transmissive to the EM used. As discussed previously, selective IRwelding effects assembly of the fluid-handling substrate whilepreventing damage to any environmentally sensitive components containedwithin the substrate.

The controllable fluid router, which may be referred to as a form of asample management engine, is operative in response to an electricalinput signal to direct fluid sample in the module to at least a selectedone of the multiple channels corresponding to the input signal. Thefluid router may direct all or part of the fluid sample to one,multiple, or all of the channels in the fluid pathway. There are manyways the router could be implemented. For example, in one embodiment therouter may consist of individual valves for each channel of the fluidpathway that are individually controlled by an electrical input signal.In another embodiment the router may perform cross routing of samplefrom one channel to another and be controlled entirely by one inputsignal. In a preferred embodiment the fluid router is a samplemanagement engine to selectively deliver and, optionally, selectivelywithdraw fluid samples from the microchannels of the probe. Preferablysamples are fed into the microchannel of one or more of the probe'smultiple detectors at a flow rate sufficiently low to maintain laminarflow. In another embodiment, the fluid routing functionality can beincorporated into one or more sample management modules. Otherembodiments will be apparent to one skilled in the art given the benefitof this disclosure.

In accordance with another embodiment the controllable router receivesthe electrical input signal from a controller unit. The controller unitmay be any number of devices including, for example, a circuit,computer, microprocessor or microcontroller. The controller unit may belocated remotely or be incorporated in the module. The controller mayoptionally be connected to various sensor units as discussed in thisspecification. The input signal delivered by the controller unit may besoftware or hardware generated. The controller may or may not be indirect or indirect communication with the NMR sites.

In accordance with another embodiment the NMR sites are in communicationwith a data processing unit. The data processing unit may be any numberof devices including, for example, a circuit, computer, ormicroprocessor. The data processing unit may be located remotely or beincorporated in the module. The data processing unit may optionally beconnected to various sensor units as discussed in this specification.The controller may or may not be in direct or indirect communicationwith the fluid router.

In some embodiments the module probe may have both the controller unitand the data processing unit. In other embodiments the controller unitand data processing unit are in communication with each other. In stillother embodiments one device, for example, a computer performs thefunctions of both. In another example, a microprocessor incorporatedinto the module performs the functions of both devices. Otherembodiments will be apparent to one skilled in the art given the benefitof this disclosure.

In accordance with another embodiment, connectivity between the NMRdetection sites and the controllable fluid router comprise a feedbackand control link. That is, sample data acquired from the NMR detectionsite is processed and fed back to the fluid router as the feedbackelement to tie the specific steps of the fluid router to the NMRdetector site. In certain preferred embodiments, data acquired from oneNMR detection site of the probe module is output by the system,optionally as one of multiple output signals to control some aspect ofthe system, especially, e.g., to determine further operation of theprobe on a sample or samples, such as whether microcoil NMR tests and/oror other action(s) or storage of the sample should be conducted or otheroperations of the probe module performed. Especially significant in thisregard is the electronic connection (e.g., by hard wire connection,wireless connection etc.) of the NMR probe to a dataprocessing/controller unit that is responsive to the signals generatedby the NMR detection site corresponding to the NMR test data generatedby the sample. The data processing/controller unit advantageously isresponsive to the signals, programmed to detect and electronicallyrecognize or identify molecular structure or analyte composition (e.g.,by automatic comparison of NMR data against a pre-stored database ofsuch data), and to act automatically on at least selected possible testresults (e.g., those pre-flagged in the database with any of severalpossible values corresponding to different follow-on actions initiatedby the controller. In accordance with preferred embodiments, the dataprocessing/controller unit is responsive to the test data signal todirect storage, disposal or movement of the sample from the current NMRdetection site to selected other NMR detection sites and/or otheranalytical sites or components of the system, e.g., by causing pumpingof volume corresponding to the length of the flow path to the othersite, and/or by controlling gates for selecting amongst multiplepossible flowpaths.

A wide array of sample management forms may be employed in operativecommunication with the probe. The probe module may operativelycommunicate with a larger system that performs sample preparation. Inone embodiment the module is connected to or part of a liquidchromatography system. In another embodiment the module is connected toor part of a capillary electrophoresis system. In another embodiment themodule is connected to or part of a dynamic filed gradient focusingsystem. In some embodiments the sample preparation may also beincorporated in the probe module as discussed in this specification. Incertain preferred embodiments wherein the channels and associated NMRmicrocoils are formed in a module, a liquid feed line (inside diameterpreferably 5 microns to 500 microns, more preferably 25 to 50,) mayconnect to a port sited in the surface of the module from any ofnumerous commercially available sample management engines can be used,such as the Capillary Liquid Chromatography system from WatersCorporation, Milford, Mass., USA. As discussed further below,capillary-LC/micro-NMR systems are especially preferred embodiments ofthe system aspect of this disclosure. Peaks and/or samples, preferablyalready conditioned, purified, concentrated, etc., coming into the probefrom the separation engine would be routed accordingly. Examples of suchsystems can be seen in FIG. 2.

In accordance with certain preferred embodiments, peaks and/or samples,optionally already conditioned, purified, concentrated, etc., are fedinto the probe from a separation engine and are routed to theappropriate NMR detection site or, optionally may be “siderailed” untillater analysis can be complete with the same coil or directed to aseparate detection coil for essentially parallel analysis. That is,samples may be “siderailed” within the multi-microcoil NMR detectionprobe, either before or after any NMR analysis has been performed on it.Analysis may be performed on the siderailed sample at that time or at alater time. Later analysis may be performed with the same microcoilalready used or the sample may be directed to a different coil foressentially parallel analysis. Any of the channels of the probe may bebranched or otherwise provide one or more stations at which a sample canbe stored. Preferred embodiments of the probe use a controllable routerfor releasably or permanently retaining a sample in a void within theNMR detection site, or other retainer or plug suitable to preventunintended loss of the sample during testing or storage. Similarly,samples can be moved externally of the probe, from the void of onechannel to that of another channel within the probe.

Other embodiments will be apparent to one skilled in the art given thebenefit of this disclosure.

In accordance with a highly innovative aspect of certain preferredembodiments of the invention, a multi-detector microcoil NMR probe asdisclosed above receives a test sample and retains it for delayed orfurther future testing. Especially in such applications as proteomicsand the like, where typically only a minute quantity of a particularanalyte is available, storage in the micro-scale NMR detection site ofthe probe module avoids unwanted additional transfers of the analyte andprovides a secure and protective storage site that readily facilitatesfuture testing. Other embodiments will be apparent to one skilled in theart given the benefit of this disclosure.

In accordance with another embodiment, a NMR probe module comprises atleast one fluid inlet port, a fluid pathway comprising multiplechannels, in fluid communication with the at least one fluid inlet port,for the transport of fluid sample to be tested; at least one operativecomponent in communication with the fluid pathway, multiple NMRdetection sites each in fluid communication with at least one of themultiple channels, each comprising as sample holding void, and anassociated RF microcoil; and a controllable fluid router operative inresponse to an electrical input signal to direct fluid sample in themodule to at least a selected one of the multiple channels correspondingto the input signal.

The operative component can be any number of devices that interact withthe fluid in the module including, for example, sensors, samplepreparation devices, pumps, heaters, coolers, ultrasonic devices andeven additional NMR sites. The operative component may also be anynumber of devices that do not interact with the fluid. Examples, forinstance, include microprocessors, micro-controllers and memory module.In some embodiments the operative component is in electricalcommunication with the controllable gate. In certain preferredembodiments the operative component is incorporated into the probe,e.g., integrated on-board the module. In other preferred embodiments theoperative component is selectively removable. Having the operativecomponent selectively removable allows for swapping of differentoperative components allowing for greater configuration flexibility. Inother embodiments the operative component is in communication with theone or more of the NMR detector sites. The operative device may alsooptionally in communication with a controller unit, a data processingunit or both. In other embodiments there may be multiple operativecomponents in any of the configuration discussed above or below, whichmay or may not be in communication with each other. Other embodimentswill be apparent to one skilled in the art given the benefit of thisdisclosure.

In one embodiment the operative component is an IR detector, aphotodiode array or the like. In still another embodiment the operativecomponent is a UV visibility array. In some embodiments the operativecomponent is an LC device. In other embodiments the operative componentis a CE device. Other embodiments will be apparent to one skilled in theart given the benefit of this disclosure.

In some embodiments the operative component is a pump. In otherembodiments the operative component is a heating device. In still otherembodiments the operative component is a sonication device. In anotherembodiment the operative component is a reaction site. Other embodimentswill be apparent to one skilled in the art given the benefit of thisdisclosure.

In embodiments with a microprocessor or micro-controller the operativecomponent may act as a controller unit, a data processing unit, or both.In embodiments where the operative device is a memory module the devicemay store data about the probe such as its configuration, when and whereit was made, etc. In other embodiments the memory module may store datafrom the NMR sites or other operative components incorporated into themodule. Other embodiments will be apparent to one skilled in the artgiven the benefit of this disclosure.

In accordance with another aspect, an NMR probe module comprises asubstrate defining at least one fluid inlet port, operative to receive afluid; fluid pathway; and multiple NMR detection cells. In certainpreferred embodiments the NMR probe module further comprises acontrollable fluid router as described above. The fluid pathwaycomprises multiple channels and is in fluid communication with the inletport. The multiple NMR detection cells are each in fluid communicationwith at least one of the multiple channels of the fluid pathway. EachNMR detection cell comprises an enlarged void for holding a fluidsample, and an associated microcoil. The controllable fluid router isoperative in response to an electrical input signal to direct fluidsample in the module to at least a selected one of the multiple channelscorresponding to the input signal. Preferably the NMR detection cellcomprises at least one RF detector microcoil associated with an enlargedvoid, that is, an enlarged sample chamber in a capillary, or channel.The inner diameter of the channel enlarges to form the enlarged void,preferably with conical tapering at each end of the void. The void mayextend axially in the channel beyond the microcoil. Optionally, one ormore of the multiple channels in the NMR probe module forms more thanone enlarged void, each with an associated NMR microcoil. In accordancewith preferred embodiments, the inner diameter of the channel on eitherside of the enlarged void is 5 um to 500 um, more preferably 25 um to 50um, the conical taper of the channel at each end of the void preferablyis at an angle to the longitudinal axis of about 5 to 75 degrees, e.g.,about 30 degrees, and the inner diameter of the enlarged void betweenthe conically tapered portions preferably is substantially uniform andfrom 100 um to 1 mm, more preferably 250 um to 800 um. The microcoil ispositioned to axially surround the void, typically being about 250 um to1 mm in the axial direction.

1. An NMR system comprising an NMR probe comprising multiple NMR detection sites, each of the multiple NMR detection sites comprising a sample holding void, and an associated NMR microcoil; and a controllable fluid router operative in response to electrical router control input signals in order to direct a fluid sample to any selected one or more of the multiple NMR detection sites; wherein at least a first plurality of the multiple NMR detection sites are arranged in fluidic series communication with each other configured for acquiring NMR spectra correspondingly in series from the fluid sample.
 2. The NMR system of claim 1 wherein each of at least a plurality of the multiple NMR detection sites comprises an NMR microcoil detector.
 3. The NMR system of claim 1 wherein the multiple NMR detection sites collectively comprise multiple NMR detectors, each of the multiple NMR detectors being operative for signal acquisition independently of others of the NMR detectors.
 4. The NMR system of claim 1 wherein the multiple NMR detection sites collectively comprise multiple transceiver NMR spectrometers.
 5. The NMR system of claim 1 wherein the multiple NMR detection sites collectively are operative to receive signals from individual ones of the NMR microcoils using time-multiplexed acquisition.
 6. The NMR system of claim 1 further comprising a controller, wherein: at least a first NMR detection site of the multiple NMR detection sites is operative to generate an NMR sample signal to the controller corresponding to an NMR spectra acquired from a fluid sample in the first NMR detection site; the controller is operative to generate a corresponding router control input signal to the controllable fluid router at least partly in response to an NMR sample signal received from the first NMR detection site; and the controllable fluid router is operative to route a fluid sample from the first NMR detection site to any of at least a plurality of the multiple NMR detection sites in response to a router control input signal from the controller generated by the controller at least partly in response to an NMR sample signal received from the first NMR detection site corresponding to the router control signal from the first NMR detection site.
 7. The NMR system of claim 6 wherein the controller comprises a microprocessor.
 8. The NMR system of claim 7 wherein the controller is programmed to respond to at least selected NMR sample signals to direct storage, disposal or movement of a fluid sample from one of the multiple NMR detection sites.
 9. The NMR system of claim 8 wherein the controller is operative to direct movement of a fluid sample from one of the multiple NMR detection sites to at least one of: others of the multiple NMR detection sites, and another component of the system.
 10. The NMR system of claim 8 wherein the controller is operative to direct movement of a fluid sample from one of the multiple NMR detection sites to another site by action comprising at least one of: causing pumping of sample volume corresponding to a length of a flow path to the other site, and controlling gates in order to select amongst multiple possible flow paths.
 11. The NMR system of claim 8 wherein the controller is operative to recognize NMR sample signals corresponding to at least one selected molecular structure or analyte composition.
 12. The NMR system of claim 8 wherein the controller is operative to recognize NMR sample signals by automatic comparison of NMR sample signals to a pre-stored database and to act automatically on at least one selected NMR sample signal.
 13. The NMR system of claim 6 wherein the sample holding void of each of the NMR detection sites is in a capillary-scale fluid channel in the probe and the controllable fluid router is operative to cross-route fluid sample from a first capillary-scale fluid channel to a second capillary-scale fluid channel in the probe in response to a router control input signal from the controller.
 14. The NMR system of claim 1 wherein the controllable fluid router is operative to direct a fluid sample in series to at least a plurality of the multiple NMR detection sites, and is operative to direct a fluid sample in parallel to at least a plurality of the multiple NMR detection sites.
 15. The NMR system of claim 1 wherein the multiple NMR detection sites are integrated with each other in a probe module.
 16. The NMR system of claim 15 wherein the probe module comprises a substrate and the microcoils are formed at peninsula extensions of the substrate.
 17. The NMR system of claim 15 wherein the sample holding void of each of the NMR detection sites is in a capillary-scale fluid channel in the probe module.
 18. The NMR system of claim 15 wherein the sample holding void of each of the NMR detection sites is in a micro-scale fluid channel in the probe module.
 19. The NMR system of claim 1 further comprising an operative component in communication with the controllable fluid router and operative to communicate signals with the controllable fluid router.
 20. The NMR system of claim 19 wherein the multiple NMR detection sites and the operative component are integrated in a probe module.
 21. The NMR system of claim 1 further comprising a controller in communication with the controllable fluid router and operative to generate the router control input signals to the controllable fluid router.
 22. The NMR system of claim 21 wherein the multiple NMR detection sites and the controller unit are integrated in a probe module.
 23. The NMR system of claim 21 wherein the controller is operative to receive information from the multiple NMR detection sites and operative to generate router control input signals to the controllable fluid router based at least in part on information from one or more of the multiple NMR detection sites.
 24. The NMR system of claim 21 further comprising an operative component, wherein the controller is operative to receive component information from the operative component and operative to generate the router control input signals to the controllable fluid router based at least in part on component information received from the operative component.
 25. The NMR system of claim 24 wherein the operative component, the controller and the multiple NMR detection sites are integrated in a probe module.
 26. The NMR system of claim 24 wherein the operative component comprises at least one of: a heating device, an IR detector, a sonication device, a UV visibility array, a photodiode array, a memory module, a pump, and a reaction site.
 27. The NMR system of claim 1 wherein the sample holding void is in a capillary-scale fluid channel.
 28. The NMR system of claim 1 wherein the sample holding void is in a micro-scale fluid channel.
 29. The NMR system of claim 1 wherein each of the multiple NMR detection sites is optimized for a different nuclear species.
 30. The NMR system of claim 1 wherein at least one of the multiple NMR detection sites is optimized for 1 dimensional NMR study.
 31. The NMR system of claim 1 wherein at least one of the multiple NMR detection sites is optimized for 2 dimensional NMR study.
 32. The NMR system of claim 1 wherein the NMR probe further comprises an analyte extraction chamber in fluid communication with at least one of the NMR detection sites.
 33. The NMR system of claim 32 wherein the analyte extraction chamber is operative to perform at least one of liquid chromatography, capillary electrophoresis, and dynamic field gradient focusing.
 34. The NMR system of claim 1 wherein at least a second plurality of the multiple NMR detection sites are arranged in parallel fluidic communication with each other. 